Abstract

Double minute chromosomes are cytogenetic manifestations of gene amplification frequently seen in cancer cells. Genes amplified on double minute chromosomes include oncogenes and multi-drug resistant genes. These genes encode proteins which contribute to cancer formation, cancer progression, and development of resistance to drugs used in cancer treatment. Elimination of double minute chromosomes, and therefore genes amplified on them, is an effective way to decrease the malignancy of cancer cells. We investigated the effectiveness of a cancer drug, gemcitabine, on the loss of double minute chromosomes from the ovarian cancer cell line UACC-1598. Gemcitabine is able to decrease the number of double minute chromosomes in cells at a 7500X lower concentration than the commonly used cancer drug hydroxyurea. Amplified genes present on the double minute chromosomes are decreased at the DNA level upon gemcitabine treatment. Gemcitabine, even at a low nanomolar concentration, is able to cause DNA damage. The selective incorporation of double minutes chromatin and γ-H2AX signals into micronuclei provides a strong link between DNA damage and the loss of double minute chromosomes from gemcitabine treated cells. Cells treated with gemcitabine also showed decreased cell growth, colony formation, and invasion. Together, our results suggest that gemcitabine is effective in decreasing double minute chromosomes and this affects the biology of ovarian cancer cells.

A. Representative pictures of metaphase spread of UACC-1598 and UACC-1598-4 cells. Arrows indicate DMs. The number of DMs in each metaphase cell was counted and plotted. ***indicates P<0.001. B. Metaphase spreads were prepared for cells grown in the presence of low concentrations of either HU or GEM for 1 week or 2 weeks. The number of DMs in each metaphase cell was counted. The red lines represent the mean and the black lines represent the SEM. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.

The signal of EIF5A2, MYCN, and MCL1 genes is decreased in HU and GEM treated UACC-1598.

A. Genes EIF5A2, MYCN, and MCL1 are present on DMs in UACC-1598 cells. Metaphase spread of UACC-1598 cells detected with DNA probe for EIF5A2, MYCN, and MCL1. For the overlapped pictures, EIF5A2 and MCL1 signals are shown in red, and MYCN signal is shown in green. Overlapped EIF5A2/MYCN and MCL1/MYCN are shown in yellow. B. Representative pictures of EIF5A2/MYCN and MCL1/MYCN FISH signals in interphase cells of UACC-1598 and how individual cells are separated into different groups. MYCN signal is shown in green, EIF5A2 and MCL1 shown in red, and the overlap is shown in yellow. The percentage of cells in Groups 1, 2, and 3 is based on EIF5A2/MYCN and MCL1/MYCN FISH signals. The statistical analysis showed the differences of cells in Group 1 and Group 2/3 compared to the control cells. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.

The amplification of genes present on DMs is decreased in UACC-1598 cells grown in the presence of HU and GEM by real-time PCR analysis.

The amplification level of genes EIF5A2, MCL1, and MYCN was analyzed by real-time PCR. The amplification level of each gene in compound treated cells is compared to control cells (DMSO for HU treated, and Ctrl. for GEM treated), and the mean relative amplification level ± SD is graphed. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01, ***denotes P<0.001.

Entrapment of EIF5A2, MYCN and MCL1 in MN in UACC-1598 ovarian cancer cell line by growth in HU and GEM.

The left two panels are representative pictures of cells showing MN without signal and the right two panels are representative pictures of cells showing MN with EIF5A2/MYCN or MCL1/MYCN signal. MYCN signal is shown in green, EIF5A2 and MCL1 are shown in red, and the overlap is shown in yellow. Arrows indicate MN.

Induction of γ-H2AX foci by HU and GEM in the UACC-1598 ovarian cancer cell line.

A. Representative picture of γ-H2AX foci detected by immunofluorescence and examples of how cells are grouped into different categories based on the degree of DNA damage. DAPI stained DNA is shown in blue and immunofluorescence of γ-H2AX foci is shown in red. B. Percentage of cells in each category based on amount of γ-H2AX foci. The statistical analysis showed the differences of cells in the No signal group vs. all other groups compared to the control cells. ***denotes a P value of <0.001.

Treatment with HU or GEM decreases the malignancy of UACC-1598 ovarian cancer cells.

A. The growth of UACC-1598 cells is decreased when grown in the presence of HU or GEM. The OD value of cells were measured every day for 6 days and plotted as mean ± SD. **denotes a P value of 0.001 to 0.01 from day 4 onwards when compared to the control group. B. Colony formation of UACC-1598 cells is decreased when grown in the presence of HU or GEM. The average numbers of colonies ± SD decreased from 365±24 colonies to 182±30 for HU treated cells and 168±25 for GEM treated cells. ***denotes a P value of <0.001 when compared to the control group. C. One representative trial of cell invasion is plotted for Ctrl., HU treated, and GEM treated cells. The invasion percentage of cells decreased from 0.58% for control group to 0.20% for HU treated cells and 0.35% for GEM treated cells. ***denotes a P value of <0.001 when compared to the control group.